Int.J.Curr.Microbiol.App.Sci (2017) 6(9):

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1 International Journal of Current Microbiology and Applied Sciences ISSN: Volume 6 Number 9 (2017) pp Journal homepage: Original Research Article Qualitative and Quantitative Analysis of Organic Acid Production Influenced by Phosphate Sources under Submerged Culture of Aspergillus niger a Phosphate Solubilising Fungi Hruda Ranjan Sahoo and Nibha Gupta * Division of Plant Pathology and Microbiology, Regional Plant Resource Centre, Bhubaneswar , Odisha, India *Corresponding author A B S T R A C T K e y w o r d s Aspergillus niger, Tricalcium Phosphate, Rock Phosphate, Organic acid. Article Info Accepted: 10 July 2017 Available Online: 10 September 2017 Aspergillus niger inoculated into medium containing Tricalcium phosphate (TCP) and Rock phosphate (RP) resulted in acid production which is indicated from the measure of ph and titrable acidity. Maximum of 105 mm acid production is recorded in TCP supplemented culture of 12 days whereas it is 124 mm in nutrient medium aided with RP and 14 days old culture. At the same time, decline of ph has also been observed in media containing TCP and RP. However, fungal biomass did not show any impact of both the kind of P sources. Chromatographic analysis of culture filtrate has showed the presence of citric and oxalic acid in TCP and RP respectively. Cultural conditions of different range of ph and incubation temperature do not play an important role in secretion by the fungi but secretion is mostly influenced by the nutritional sources such as carbon and nitrogenous compounds present in the medium. Introduction Organic acids are the source of biotical generated H + ions, which can mineralize bound phosphate present in soil and make it available for the uptake by plants (Bhattacharya and Jain, 2000).The organic acid concentration in soil solution is typically low, varying from 1 to 50 mm (Strobel, 2001). Hence, microbial production of low molecular weight s enhances mobilisation of phosphate compounds (Bolan et al., 1994; Goldstein, 1995) which is also accompanied by acidification of the medium. Organic acids such as acetate, citrate, gluconate, lactate, malate, oxalate, succinate 3358 etc. can form complexes with the metals such as aluminium, calcium, and iron releasing phosphate into soil from respective metal phosphates andcan also increase P availability by blocking P adsorption sites on soil particles (Jones, 1998; Rodriguez and Fraga, 1999; Gyaneswar et al., 2002). It is observed that tri- and dicarboxylic acids are more effective in solubilising P as compared to monobasic and aromatic acids aliphatic acids (Mahidi et al., 2011).The solubilization of different sources of P does not depend only on the total amount of acids produced but also on type of acid produced (Cunningham and

2 Kuiack, 1992, Kang et al., 2002). However, production is also influenced by the micro-organism and P source where it grows (Scervino et al., 2010). The type of produced and their amounts may also differ with different organisms. A number of factors have been considered as they also affect the production with ph lowering by microorganisms, such as the C and N sources (Di Simine et al., 1998; Reyes et al., 1999). Aspergillus niger is a fungus which is known to solubilise inorganic phosphate such as calcium and aluminium phosphates (Illmer et al., 1995; Barroso and Nahas; 2005) through production of s such as citric, gluconic, succinic and oxalic acid along with drop in ph of the medium (Nahas et al., 1990; Illmer and Schinner, 1995). Studies by Venketeswarlu et al., 1984 reported lactic acid as only acid produced by A. niger while Vazquez et al., 2000 reported succinic acid as the only acid produced by A. niger. Reports also reveal that carbon as well as nitrogen sources and ph of the medium greatly influence production (Gupta et al., 1976). In the present study, we have conducted experiments to observe the production at periodic intervals when the organism A. niger is inoculated with different phosphate sources such as Tricalcium phosphate and rock phosphate as well as on modification of culture conditions such as temperature, ph and nutritional parameters such as carbon and nitrogen sources in presence of TCP and rock phosphate. Materials and Methods Modified Czapekdox medium containing Tricalcium phosphate (ph 6.8) and rock phosphate (ph 7.2) were inoculated with equal amount of inoculum of Aspergillus niger and incubated at 28ºC for a period of 2 weeks. At a regular interval of 48 hours from period of incubation, flasks proliferated by the organism were analysed for their ph and titrable acidity. The ph change of the culture medium was recorded with the help of ph meter. Titrable acidity (TA) was estimated using or titrating 1ml of culture supernatant against 10 mm NaOH in presence of phenolphthalein indicator at regular interval (Whitelaw et al., 1999). The biomass obtained was air dried and weighed. The culture supernatant obtained was concentrated and spotted on the chromatographic paper along with the standards of s. The chromatogram was run in pre saturated chamber containing solvent mixture of n-butanol, acetic acid and water in the ratio 12:3:5. The chromatogram was air dried and sprayed with bromocresol green. After air dried the Rf values of the yellow spots of s developed on blue background was measured and compared with Rf values of standard for identification. The same Czapekdox Medium was prepared with further modification of carbon and nitrogen sources (nutritional parameters) and ph and temperature (Cultural parameters) for estimation of titrable acidity and recording of ph after completion of incubation period.for studies with nutritional modification, the carbon sources such as Fructose, Glucose, Inositol, Lactose, Maltose, Mannose, Raffinose, Sorbose and sucrose were taken in the medium and ph maintained at 7 and temperature 28 C. Likewise, nitrogen sources such as Ammonium chloride, Ammonium Sulphate, L-Glutamine, L-Phenylalanine, L- Threonine, L-valine, Potassium nitrate, Urea and Sodium nitratewere taken in the medium and ph maintained at 7 and temperature 28 C. Similarly for studies with cultural modifications, the ph of the medium was 3359

3 modified in the range of 5-9 and incubated at constant temperature of 28 C. The incubation temperature was modified in the range of C with ph of the medium at 7. The amount of produced and change in ph of the medium in all the modifications was recorded. Results and Discussion The titrable acidity ranged from mm s during the incubation period of 2 weeks except for 12 th day where it is maximum (105 mm) in case of solubilisation of TCP. Results obtained from studies by Nenwani et al., 2010 also showed that highest titrable acidity is recorded after 12 days of incubation by the organism which is similar to our findings. However, there is increase in the titrable acidity during the entire period of incubation when Aspergillus niger is inoculated into the medium containing rock phosphate and maximum of 124 mm of total acids is produced at the end of 14 th day of incubation as shown in table 1. A significant correlation between final ph value and titrable acidity has been observed (Nahas, 1996). When the fungal strain was tested to solubilise different types of phosphate in chemosynthetic medium, medium inoculated with A. niger showed greater reduction in ph (Reena et al., 2013). There was decrease in the ph of the medium ranging from 2-3 both in presence of TCP and rock phosphate except for 12 th day in case of TCP and 8 th day in case of rock phosphate where the ph is recorded as 3.74 and 3.89 respectively. The pattern of decrease in ph is similar in case of both TCP and rock phosphate supplemented medium by A. niger. The biomass obtained due to the proliferation of the organism during the incubation period is recorded. At the end of 14 th day, the biomass obtained from both the phosphate sources is equal (0.174g) which depicts that the organism is able to grow unanimously irrespective of the phosphate sources present in the medium either TCP or rock phosphate. A strong positive correlation of is observed between ph and total acids secreted by A. niger during TCP solubilisation which is 0.49 in case of rock phosphate solubilisation. This proves that production is the key mechanism for P solubilisation which is accompanied by lowering of the ph of the medium. This fact is confirmed that production of s leads to acidification of microbial cells and their surroundings resulting in the release of orthophosphate ions from the mineral phosphate (Hwangbo et al., 2003; Ben et al., 2009). It has been observed that fungal culture produced carboxylic acid confirmed as titrable acidity. The culture supernatant obtained was spotted on whatman chromatographic paper No.1 along with the standards of s. The Rf values obtained for the culture supernatant obtained from Aspergillus niger inoculated with TCP and rock phosphate were 0.9 and 0.6 respectively when compared to the standard s it coincided with oxalic acid (0.91) and citric acid (0.63) respectively (Figure 1). Similarly, Illmer and Schinner, 1995 and Alam et al., 2002 also reported citric and oxalic acid to be produced by Aspergillus niger in large amounts along with small quantities of gluconic acid. Modification of cultural and nutritional conditions also influences the production by the organism in presence of TCP and Rock phosphate. Nutritional modifications involve replacement of carbon and nitrogen sources in the medium. In presence of TCP, all the carbon sources were effectively utilised by the organism in order to produce in the medium except lactose, sorbose and inositol since much 3360

4 reduction in the ph of the culture filtrate is not observed in presence of these three carbon sources (Figure 2a). Similar trend is also noticed in case of secretion which is reflected from the measure of titrable acidity and maximum titrable acidity during TCP solubilisation is recorded in presence of glucose (127mM) followed by sucrose (116 mm). However, in presence of rock phosphate; all the carbon sources were taken up by Aspergillus niger as energy source which is indicated from the amount of organic acid present in the medium except mannose. However, amount of titrable acidity is recorded higher in glucose (45mM) followed by maltose and sucrose (34 and 32 mm). Not much change in ph is observed in presence of mannose but other carbon sources showed ph in the range of except fructose and raffinose as depicted from figure 2b. Initial ph of the medium Incubation period Table.1 Periodical analysis of Biomass, and change in ph of culture filtrate of A. niger grown under different phosphate sources production in culture filtrate (mm) Tricalcium Phosphate Final ph of culture Fungal biomass filtrate (in g) production in culture filtrate (mm) Rock Phosphate Final ph of Fungal culture biomass filtrate (in g) Control Day Day Day Day Day Day Day Table.2 Effect of ph of the culture medium on production (titrable acidity) in presence of TCP and RP production in culture filtrate (mm) Tricalcium Phosphate Final ph of culture filtrate Fungal biomass (in g) production in culture filtrate (mm) Rock Phosphate Final ph of culture filtrate Fungal biomass (in g) 5 53±1 3.68± ± ±4 4.25± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0 3.97± ± ± ± ± ±2 4.98± ± ± ± ±

5 Table.3 Effect of Temperature of the culture medium on production (titrable acidity) in presence of TCP and RP Tricalcium Phosphate Rock Phosphate Final ph of Final ph Temperature ( C) production in culture filtrate (mm) culture filtrate Fungal biomass (in g) production in culture filtrate (mm) of culture filtrate Fungal biomass (in g) 20 41± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2 3.97± ± ± ± ±0.009 Fig.1 Qualitative analysis of s produced by Aspergillus nigerunder different phosphate sources (Rf values) Fig.2a Organic acid secretion in form of titrable acidity in presence of TCP and RP supplemented with different carbon sources Amount of secreted (in mm) TCP RP Carbon sources 3362

6 Fig.2b ph change during production supplemented with different carbon sources in presence of TCP and RP Change in ph of culture filtrate TCP RP Carbon sources Fig.3a Organic acid secretion in form of titrable acidity in presence of TCP and RP supplemented with different nitrogen sources 3363

7 Fig.3b ph change during production supplemented with different nitrogen sources in presence of TCP and RP For nitrogen sources, all the nitrogenous compounds and amino acids were suitably consumed by the organism for its metabolic activity since the amount of T.A. was higher in presence of TCP for all the nitrogen containing compounds with glucose as carbon source in the range of mm suggesting that modification enhanced the producing ability of A. niger as shown in figure 3a. Maximum titrable acidity was recorded in medium modified with amino acid L-valine (179 mm). Similar observation was noted for ph change in the culture filtrate with Final ph ranging from in presence of TCP and with rock phosphate supplementation in the medium in presence of different nitrogen sources it was noticed that there is greater variation in the ph of the culture filtrate with wider ph ranging from (Figure 3b). The titrable acidity estimated is the amount of secreted into the medium was found to be lower than that of TCP supplemented medium and highest T.A. was indicated in presence of potassium nitrate (49 mm) followed by sodium nitrate (39 mm). Upon modification of the initial ph of the medium, the change in the final ph of the medium in presence of TCP as P source varied from 3.68 (ph 5) to 4.98 (ph 9) and amount of titrable acidity also ranged from mm under such conditions. The ph change at different incubation temperatures also ranged from 3.44 (25 ºC) to 3.97 (40ºC) which suggested that is secreted into medium by lowering of ph and at ambient temperature of incubation. However, the amount of acid release at different temperature range is almost same (41-47 mm) as much difference in their values is not observed. In presence of rock phosphate, the ph change is not much significant because the final ph for all modifications is in the range of Similar findings were also recorded in case of various incubation temperatures for rock phosphate also as the final ph ranged from The amount of titrable acidity measured at different ph and temperature conditions showed that it is more in case of initial ph 6 and temperature 20ºC which is 37±2.3mM and 36±1.73 mm respectively (Tables 2 and 3). 3364

8 Overall, it can be concluded that cultural conditions of different range of ph and incubation temperature do not play an important role in secretion by the fungi. Hence, the secretion is mostly influenced by the nutritional sources present in the medium which regulates the metabolic pathway of the organism. Although production influences the solubilisation process as determined from ph and titrable acidity in the present in vitro study but in vivo effect on the plant growth can only justify the potential of the strain used in this experiment. Acknowledgement The financial assistance obtained through INSPIRE programme (No. DST/INSPIRE Fellowship/2013/506) DST, Govt. of India is gratefully acknowledged. References Alam, S., Khalil, S., Ayub, N. and Rashid, M In vitro solubilisation of Inorganic phosphate by Phosphate Solubilising Micro-Organisms (PSM) from maize rhizosphere. International journal of Agriculture and biology. 4(4): Barroso, C.B., Nahas, E The status of soil phosphate fractions and the ability of fungi to dissolve hardly soluble phosphates. Appl. Soil Ecol. 29, Ben Farhat, M., Farhat, A., Bejar, W., Kammoun, R., Bouchaala, K., Bejar, S., Chouayekh, H Characterization of the mineral phosphate solubilizing activity of Serratia marcescens CTM isolated from the phosphate mine of Gafsa. Arch Microbiol. 191, Bhattacharya, P., Jain, RK Phosphorous solubilizing biofertilizers in the whirl pool of rock phosphate - challenges and opportunities. Fert News. 45, Bolan, N.S., Mahimairaja, S., Baskaran, S Influence of low molecularweight s on the solubilization of phosphates. Biol. Fertil. Soils, 18, Cunningham, J.E., Kuiack, C Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii. Appl. Environ. Microbiol. 58, Di Simine, C.D., Sayer, J.A.; Gadd, G.M Solubilization of zinc phosphate by a strain of Pseudomonas fluorescens isolated from a forest soil. Biol. Fertil. Soils, 28, Goldstein, A.H., Recent progress in understanding the molecular genetics and biochemistry of calcium phosphate solubilization by Gram negative bacteria. BiolAgricHortic 12: Gupta, J.K., Heding, L.G.; Jorgensen, O.B Effect of sugars, hydrogen ion concentration and ammonium nitrate on the formation of citric acid by Aspergillus niger. ActaMicrobiol. Acad. Sci. Hun.,23, Gyaneshwar, P., Kumar, N., Parekh, L.J., Poole, P.S Role of soil microorganisms in improving P nutrition of plants. Plant Soil 245: Hwangbo, H., Park, R.D., Kim, Y.W., Rim, Y.S., Park, K.H., Kim, T.H., Suh, J.S., Kim, K.Y ketogluconic acid production and phosphate solubilization by Enterobacter intermedium. Curr. Microbiol. 47: Illmer, P., and Schinner, F Solubilization of inorganic calcium phosphate - solubilization mechanisms. Soil Biol. Biochem. 27: Illmer, P., Barbato, A., Schinner, F Solubilization of hardly solublealpo4 3365

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